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ANIMAL NUTRITION |
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* Department of Animal Science, Universidade Federal de Viçosa, Viçosa, MG 36571, Brazil; and
Department of Animal Science, Texas A&M University, College Station, TX 77843-2471
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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0.28) in microbial protein synthesis and microbial efficiency among the treatments. The results of these trials suggest that dietary NPN levels (up to 46.5% of total N) can be fed to crossbred steers receiving corn silage-based diets without affecting their growth performance or ruminal protein synthesis.
Key Words: feedlot • nonprotein nitrogen • protein supplementation
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Consequently, asynchrony of N and digestible energy occurs because of rapid N release from urea, and excess ammonia will not be efficiently used.
According to early studies, urea can be effectively utilized when dietary inclusion is limited to one-third of supplemental N or 1% of dietary DM (Reid, 1953; Chalupa, 1968). In contrast, other studies (Rennó et al., 2005; Magalhães et al., 2006) have demonstrated that intake and performance were not affected when high urea levels (1.95% of dietary DM, approximately 46% of total N as NPN, from urea/ammonium sulfate) were added in the diet or when supplemental true protein was replaced with urea. However, few experiments have been designed to identify the amount of dietary NPN needed for maximum animal performance, ruminal fermentation, and MP supply in cattle consuming corn silage-based diets. Utilization of the correct levels of dietary NPN required for optimum N use by ruminal microbes would allow adequate performance, thereby improving feed efficiency and reducing feed costs and N losses to the environment.
The objectives of this study were to evaluate the effects of dietary NPN levels in steers consuming corn silage-based diets on intake, growth, nutrient digestion, microbial protein synthesis, and ruminal characteristics.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Location and Diets
The experiments were conducted at the Federal University of Viçosa, Brazil, during April to July of 2004. Diets were the same for Exp. 1 and Exp. 2, and were formulated to provide increasing levels of dietary NPN. Treatments consisted of 0, 15.5, 31, and 46.5% of dietary N as NPN (Table 1). The 2 sources of added NPN were urea and ammonium sulfate. In the treatment diets, urea, ammonium sulfate, and ground corn replaced cottonseed meal of the control diet. Diets consisted of 70% corn silage and 30% concentrate (DM basis), formulated to contain 12.5% CP (DM basis).
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Steers and Sampling Protocol. Twenty-four Nellore x Holstein crossbred steers (350 ± 20 kg of BW) were distributed in 6 blocks to evaluate intake and digestibility of nutrients and performance of the steers in the feedlot. Steers were blocked into 6 groups based on initial BW and allotted randomly to 1 of 4 treatments (6 steers per treatment). The steers were treated for internal and external parasites at the beginning of the experiment and kept in individual pens of approximately 10 m2, with protected feeders and water. The experiment was conducted for 99 d (15 d for diet adaptation, and 3 periods of 28 d for data collection).
Steers were individually fed for ad libitum intake twice daily at 0700 and 1500 h. Diets were fed as a total mixed ration, in which corn silage and concentrate (previously mixed) were weighed and mixed before feeding. Orts were collected and weighed once daily, and diets were adjusted daily to yield orts of approximately 5 to 10% of the total feed offered. Steers had free access to water. Feed ingredients and orts were sampled daily and composited by weight within a period.
For each animal, DMI was measured daily and grab samples of feces (approximately 200 g) were collected between d 42 and 44, with collection intervals of 28 h. Indigestible ADF (iADF) was used as an internal marker to estimate apparent nutrient digestibility and fecal output. After drying at 60°C for 72 h, feed, ort, and fecal samples were ground to pass a 1-mm screen (Wiley mill, model 3, Arthur H. Thomas, Philadelphia, PA), and period composites per steer were prepared. Composite samples were used to determine DM (method 934.01; AOAC, 1990); OM determined by ash (method 924.05; AOAC, 1990); CP obtained by total N determination using the micro-Kjeldahl technique (method 920.87; AOAC, 1990) and a fixed conversion factor (6.25); ether extract (EE) determined gravimetrically after extraction using petroleum ether in a Soxhlet instrument (method 920.85; AOAC, 1990); NDF (Van Soest et al., 1991); ADF (method 973.18; AOAC, 1990); and sulfuric acid lignin (Robertson and Van Soest, 1981). The NDF and ADF were not corrected for ash or protein. The iADF (ADF remaining after a 144-h in situ incubation in a rumen-cannulated cow) was determined according to the method of Cochran et al. (1986), and the digestibility of nutrients was calculated as
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Nonfiber carbohydrate (NFC; %) was calculated by difference as:
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except for diets containing urea and ammonium sulfate, where NFC was calculated as (Hall, 2000):
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Apparent TDN (%) was calculated as (NRC, 2001):
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The NPN percentage of feed samples was determined through precipitation of true protein by trichloroacetic acid and subsequent filtration and determination of the insoluble N in the residue (Licitra et al., 1996). The NPN was calculated as the difference between total N and precipitated true protein N.
Animal Performance. The ADG was calculated as the difference between the final and initial shrunk BW (16 h of fasting, only water was provided) divided by the number of days of feeding (84 d). Gain efficiency was calculated as ADG divided by DMI.
Exp. 2
Steers and Sampling Protocol. Four Holstein x Nellore steers (300 kg ± 55 kg of BW) fitted with abomasal and ruminal cannulas were used in a 4 x 4 Latin square design to evaluate dietary intake and apparent total tract and partial digestibilities of nutrients, ruminal pH, and ammonia concentration, rumen microbial protein synthesis and plasma urea. Each experimental period was 19 d: 10 d for adaptation to the diet, 6 d to collect fecal and abomasal samples, 1 d for ruminal pH measurements and collection of ruminal fluid samples, 1 d for blood collection, and 1 d to collect ruminal contents to isolate bacteria. The experiment was conducted for 76 d (4 periods of 19 d each).
Steers were assigned randomly to 4 dietary treatment sequences and were fed individually for ad libitum intake twice daily (0700 and 1500 h). Diets were fed as total mixed rations, as described for Exp. 1. Orts were collected and weighed once daily, and feed offered was adjusted daily to yield orts of approximately 5 to 10% of the total feed offered. Steers had free access to water. Feed ingredients and orts were sampled daily and composited by weight for each steer within each period.
Feces and abomasal digesta samples (approximately 200 g and 500 mL, respectively) were collected between d 11 and 16 of each period, with intervals of 26 h between the samplings. Abomasal fluid subsamples were preserved with 1 mL of 9 M H2SO4, and stored at –20°C for analysis of NH3. Abomasal fluid NH3 was analyzed by distilling with 2 M KOH in a micro-Kjeldahl system, after previous centrifugation at 1,000 x g for 15 min, according to the original procedures of Fenner (1965) and the adaptations of Vieira (1980).
Fecal and abomasal samples were dried in a forced-draft oven (60°C for 72 h) and then ground to pass a 1-mm screen. Fecal and abomasal samples were composited on a DM basis to obtain representative composite samples for each steer within each period. Composite samples of feeds, orts, feces, and abomasal digesta were analyzed for total N, DM, ash, OM, EE, NDF, and iADF, as described for Exp. 1.
Ruminal contents (100 mL) were obtained at 0, 2, 4, 6, and 8 h after the morning feeding on d 17 of each period and were subsequently strained through 2 layers of cheesecloth. The pH was measured immediately. The ruminal fluid was preserved by addition of 1 mL of 9 M H2SO4, and stored at –20°C for analysis of NH3. Ruminal fluid NH3 was analyzed as described for abomasal samples.
On d 18 of the experimental period and 4 h after feeding, blood was collected from the coccygeal artery or vein of each steer into blood collection tubes containing heparin [Vacuette, Greiner Bio-One (Americana, SP, Brazil)]. All samples were placed on ice, centrifuged at 1,500 x g for 15 min to obtain plasma, and frozen at –20°C until analysis of urea by an enzymatic-colorimetric technique using a commercial kit (Uréia CE, Labtest Diagnóstica S.A., Lagoa Santa, Brazil), which is based on the urease reaction according to Kerscher and Ziegenhorn (1985).
Determination of Microbial Protein Synthesis.
On d 19, the ruminal contents were obtained 4 h postfeeding and squeezed through 2 layers of cheesecloth to yield approximately 1.5 L of strained fluid. Particles retained on the cheesecloth were mixed with 500 mL of NaCl (9 g of NaCL/L), blended for 1 min, refiltered through cheesecloth, and added to the 1.5-L ruminal fluid sample. Bacteria were isolated by differential centrifugation (500 x g and 27,000 x g), according to procedures of Cecava et al. (1990). The resulting bacterial pellets were dried at 60°C for 48 h and ground in a ball mill (TE350, Tecnal, Piracicaba, Brazil). The ground bacterial samples were analyzed for DM, ash, and total N according to the procedures described earlier, and total purines were determined (Ushida et al., 1985). To quantify microbial protein and subsequently determine microbial efficiency, approximately 400 mg of dry abomasal digesta was used, which also was analyzed for purines according to the methods of Ushida et al. (1985).
Statistical Analyses
Exp. 1. Intake, digestibility, and performance were analyzed as a randomized complete block design using the MIXED procedure (SAS Inst. Inc., Cary, NC). The model included the fixed effects of treatment and the random effects of block. Linear, quadratic, and cubic effects of dietary NPN were tested using orthogonal contrasts. The statistical model was
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where Yijk was the measured variable, µ was the overall mean, bi was the random effect of the ith block, tj was the fixed effect of the jth treatment, and eijk was the residual error. Treatment differences were considered to be significant when P 
0.05.
Exp. 2.
The data for digestibility, intake, plasma N, abomasal N flow, and microbial protein efficiencies were analyzed as a 4 x 4 Latin Square design (Kuehl, 2000) using the MIXED procedure of SAS, and the statistical model was
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where Yijk was the variable measured, µ was the overall mean, 
i was the fixed effect of the ith treatment, bj was the random effect of the jth animal, pk was the random effect of the kth period, and eijk was the random effect of the ith treatment, jth animal, and kth period. Linear, quadratic, and cubic effects of NPN levels were tested using orthogonal contrasts. Treatment differences were considered to be significant when P 0.05 and were considered to indicate a trend at 0.05 < P < 0.10.
The ruminal characteristics data collected over time were analyzed as repeated measures (Kuehl, 2000) using the MIXED procedure of SAS. Model effects in the whole plot were animal, period, and treatment, whereas subplot effects were sampling time and treatment x sampling time interactions:
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where Yijklm was the dependent variable, µ was the overall mean, i was the fixed effect of the ith treatment, bj was the random effect of the jth animal, pk was the random effect of the kth period, 
ijk was whole plot error, zl was the effect of time, zli was the interaction between time and treatment, and 
ijklm was the subplot error. When treatment interacted (P 0.05) with sampling time, the variables were analyzed within time periods. The variance-covariance structure AR(1) was used for estimating the covariances. Differences were considered to be significant when P 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Diets (Table 1
) provided similar amounts of DM, OM, CP, EE, and NDF to all steers. Calculated ruminally degraded protein (RDP) and NPN percentages increased as urea and ammonium sulfate were added to the diets, creating a variation in the amount of available N in the rumen that was received by steers as the protein supplement. Additionally, as cottonseed meal was replaced by urea and ammonium sulfate, ground corn was also added to the diets increasing dietary NFC.
Feed Intake and Performance
There were no differences (P > 0.05) in the daily intakes of DM, OM, CP, NFC, and TDN among treatments in Exp. 1 (Table 2). The NDF intake decreased linearly (P = 0.01) as dietary NPN increased due to lesser dietary NDF with more NPN (Table 1) and a numerical decrease in DMI. In Exp. 2, there were differences (P 0.02) among the treatments only in NFC and EE intakes (Table 3). The NFC intake increased linearly (P = 0.02) with NPN due to a higher NFC percentage in those diets with more NPN, which contained greater amounts of ground corn (Table 1), and there was a quadratic effect (P = 0.05) on DMI (% of BW) as NPN increased. There are many studies with contrasting results regarding effects of urea levels (NPN) in cattle diets on DMI. Rennó et al. (2005) observed no effect of dietary urea (0 to 1.95%) replacing soybean meal on intake of nutrients in steers of 4 different genetic groups fed diets containing 50% bermudagrass hay. Similarly, Souza (2004) evaluated the effects of dietary urea (0, 0.5, 1.0, or 1.5%) on intake and performance in steers fed diets containing 70% sorghum silage and reported no differences on intake of nutrients. On the other hand, Milton et al. (1997) observed a cubic effect on DMI, which was smaller for steers consuming 0.5 and 1.5% urea than for those fed 0 and 1.0% urea in 90% concentrate diets. However, these authors reported that DMI responded quadratically to dietary urea level (0, 0.35, 0.70, 1.05, and 1.40%, DM basis); the maximum DMI was observed in steers consuming 1.05% urea and a decrease of 0.7 kg/d of DMI was observed with 1.40% urea.
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). These results are consistent with nutrient intake data because no differences were found between intakes of the majority of dietary nutrients. Results similar to ours were reported by Souza (2004), who found no differences in ADG among steers fed diets containing 70% sorghum silage and urea up to 1.5% of DM. Similarly, Magalhães et al. (2006) found no differences in ADG when they fed steers with different levels of urea (up to 1.95%, DM) replacing soybean meal in diets based on corn (70% DM) with grass silage as the roughage source. Paixão et al. (2006) replaced soybean meal with urea and reported no differences in ADG of steers fed diets containing 2 different concentrate levels (0.75 or 1.25% of BW daily). Gleghorn et al. (2004) evaluated effects of CP concentration (11.5, 13, and 14% of dietary CP) and degradability (100% urea and 0% cottonseed meal; 50% urea and 50% cottonseed meal; and 100% cottonseed meal) on the performance of beef steers fed 90% concentrate diets, and no differences in ADG were observed among CP sources. In our experiment, the ratio of NFC:RDP was similar among the diets (4.8, 4.9, 4.7, and 4.7 for diets with 0, 15.5, 31, and 46.5% NPN). The replacement of cottonseed meal with urea and ammonium sulfate increased dietary RDP. This replacement provided more NFC to the diets because ground corn was added with urea and ammonium sulfate. Because cottonseed meal has little starch and ground corn has lots of starch, all diets provided enough energy to use the RDP, and the NFC:RDP ratio was similar. Furthermore, the lack of differences for intakes of the majority of nutrients as well as animal performance possibly was due to this similar ratio of NFC:RDP.
Diet Digestibility
In Exp. 1, no effects (P > 0.05) of NPN levels were observed on apparent total tract digestibility of DM, OM, NDF, NFC, and on dietary TDN (Table 4), which were on average, 70.1, 71.3, 54.0, 86.8, and 70.9%, respectively. The linear increase in the CP apparent total tract digestibility with increasing levels of dietary NPN is likely due to greater absorption of NPN as ammonia than digestibility and absorption of cottonseed meal N.
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). The apparent digestibility of CP showed a positive linear tendency (P = 0.09), and apparent digestibility of NFC had a quadratic behavior (P = 0.04) as NPN levels increased. Rennó et al. (2005) reported no differences among levels of dietary urea (up to 46% of total N as NPN) on total tract digestibility of several nutrients in steers fed bermudagrass hay. Accordingly, there were no differences (P > 0.05) among the treatments on the percentage of total tract digestion that occurred in the rumen for DM, OM, EE, NDF, and NFC (Table 6). As expected, urea level linearly increased (P = 0.01) the proportion of CP digestion that occurred in the rumen, likely because of absorption of NPN as ammonia before the abomasum. A similar result was reported by Milton et al. (1997) who observed linear increases in steers in apparent ruminal N digestion as urea levels increased up to 1.5%, although in that study, dietary N concentration increased with urea supplementation, and this could have accounted for the increased ruminal N digestion. Furthermore, these authors observed that all diets led to negative apparent ruminal N digestion, indicating a large amount of N being recycled. In our trial, ruminal N digestibilities were positive, reflecting the excess of RDP for all diets. Although our diets had similar ratios of NFC:RDP, the percentage of NPN was different, and this likely resulted in faster release of ammonia than energy in diets with more NPN, which would lead to a surplus of N or a possible asynchrony between ruminal availability of energy and N. Therefore, these results suggested an imbalance over time between available energy and N and consequently a positive apparent CP ruminal digestibility due to the ruminal absorption of the N excess.
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There were no effects of NPN level (P = 0.14) and treatment by sampling time interactions (P = 0.58) on ruminal pH. The overall mean ruminal pH was 6.03, which is greater than the 5.0 to 5.5 range that was suggested by Hoover (1986) in which ruminal digestibility of fiber is negatively affected. In fact, the apparent ruminal digestibility of NDF was not affected by the variation of NPN levels in the diets (Table 6).
As expected, the ruminal NH3-N concentration was affected by interaction of sampling times by treatment (P = 0.013), by treatment (P < 0.0001), and by time after feeding (P < 0.0001). The sampling time by treatment interactions (Figure 1) showed a quadratic behavior with maximum ammonia (predicted by fitting quadratic equations to the data and solving for the maximum point) of 14.6, 19.5, 18.1, and 25.6 mM at 3.4, 3.5, 3.1, and 3.8 h after feeding for diets with 0, 15.5, 31, and 46.5% NPN, respectively. During all sampling times the ruminal NH3-N concentrations were well above levels (3.57 mM) recommended to optimize ruminal digestion (Satter and Slyter, 1974), although the concentration of ruminal NH3 necessary for optimal ruminal digestion on various diets is not well defined yet. Nevertheless, these results suggested that the increase of supplementation with NPN resulted in an accumulation of ruminal NH3, indicating that microbial requirements for NH3 were exceeded, or ruminal microbes were not able to utilize the N either because energy was first limiting or microbial growth was slower than the solubilization of N. Paixão et al. (2007) observed that ruminal NH3 concentration increased when soybean meal was replaced with urea. On the other hand, Souza (2004) replaced soybean meal with different levels of urea (0, 0.5, 1, and 1.5%, DM) in growing steers and found no differences on ruminal NH3.
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0.05), whereas NH3-N flow increased linearly (P = 0.003). The increase of NH3 flow probably occurred because of NPN excess in the rumen, contributing to a greater concentration in the abomasum. On the other hand, the total N and NAN flow to the abomasum decreased due to less ruminally undegraded protein in the diets with increasing levels of NPN.
Plasma urea concentrations increased linearly (P = 0.01) with addition of NPN in the diets. During periods of excessive N availability in the rumen, NH3 is absorbed and appears as urea in the plasma urea pool (Cocimano and Leng, 1967). This N may then be recycled back to the rumen or excreted in the urine. Our results for ruminal NH3 and plasma urea presumably reflect the less efficient utilization of total N that results from an excessive supply of NPN. This excessive supply of NPN would lead to a surplus of ammonia in the rumen that occurred in this trial, increasing hepatic urea synthesis. These results are in agreement with previous observations by Rennó (2003) and Magalhães et al. (2005), who used diets similar to ours and reported that plasma urea was increased when NPN (urea) was added to the diets. Cecava and Hancock (1994) also determined that urinary N and plasma urea were greater for steers fed 60% corn silage-based diets with urea than steers receiving combinations of soybean meal and feather meal. In contrast, Knaus et al. (2001) found no differences on plasma urea N in steers fed 85% concentrate diets with soybean meal or urea (1.8% of DM) as the only protein supplement.
Ruminal Fermentation and Microbial Efficiency
Dietary NPN levels had a quadratic effect (P = 0.03) on ruminally degraded OM, whereas ruminally degraded carbohydrate only showed a tendency (P = 0.06) to be quadratically affected by NPN level (Table 7). Bacterial growth is largely dependent on the amount of ammonia and fermentable OM available in the rumen (Bryant and Robinson, 1962). Although rumen-degraded OM changed quadratically as dietary levels of NPN increased and ruminal NH3 was not limiting, microbial N production (Table 7) was not affected by treatment. Moreover, microbial efficiency was not influenced by dietary NPN (P > 0.05) when expressed on a rumen-degraded OM, rumen-degraded carbohydrate, or TDN basis. These results suggested that all diets provided adequate amounts of ruminally available N for microbial protein production. If a deficiency of available amino acids and peptides occurred due to the addition of urea, microbial growth was likely not limited.
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evaluated the influence of urea supplementation (0, 0.4, 0.8, and 1.2%, DM basis) on digestive function of cattle and observed no effect on flow of rumen microbial N to the small intestine. Indeed, microbial efficiency (g of microbial N/kg of truly fermented OM) decreased linearly with increasing urea level. Milton et al. (1997) observed that flows of total and microbial N to the duodenum and microbial efficiency were not affected by dietary urea. Similarly, Paixão et al. (2006) observed no effects of replacing soybean meal by urea on microbial efficiency in steers fed diets with 2 levels of concentrate (0.75 and 1.25% of BW daily). Our findings suggest that dietary NPN (up to 46.5% of total N) can be fed to crossbred steers receiving corn silage-based diets without affecting their growing performance and ruminal protein synthesis. In addition, cottonseed meal might be replaced by urea (NPN source) in corn silage-based diets for crossbred steers and allow for ADG of 1 kg/d. However, feeding excess NPN will not improve animal performance and may cause excessive nitrogen excretion and ammonia volatilization into the environment. Nevertheless, the recommended level of dietary NPN depends on the criteria used to define optimum N utilization because NPN levels required to reduce diet costs and to maximize performance and microbial efficiency may not match those required to minimize environmental N load.
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Footnotes |
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2 Corresponding author: luis.tedeschi{at}tamu.edu
Received for publication September 25, 2006. Accepted for publication February 1, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
NH3 = –0.2786x2 + 1.896x + 11.47; R2 = 0.37; SE = 4.3; 
NH3 = –0.5775x2 + 4.079x + 12.28; R2 = 0.82; SE = 4.9; 
NH3 = –0.4091x2 + 2.518x + 14.22; R2 = 0.70; SE = 5.0; x NH3 = –0.8819x2 + 6.734x + 12.72; R2 = 0.94; SE = 7.4.